This project will address the therapy of mesothelin expressing tumors by developing and testing engineered T cells with potent antitumor cytotoxicity. Mesothelin is a tumor-associated antigen that is frequently over expressed on mesothelioma, non-small cell lung cancer, pancreatic and ovarian cancers. Our strategy is the "T-body" approach, which uses genetically reprogrammed, patient-derived lymphocytes transfected with a novel chimeric receptor that contains combinations of the signal transduction domains of 4-1BB (CD137), CD28, and CD3<; as well as anti-mesothelin scFv (anti-meso-CD28-41BB-Q. The central hypothesis that we wish to test is that insufficient numbers of CTL with adequate engraftment, persistence and effector function to self antigens have been used in previous trials of adoptive therapy for cancer. Presently, we are the only laboratory in the world that is actively testing lentiviral modified T cells in the clinic, and in that trial we have demonstrated safety and prolonged lentiviral gene transfer. The following three specific aims will test the hypothesis that engineered human T cells expressing an anti-mesothelin-CD28-41BB-<; chimeric receptor will have potent antitumor activity in vitro and in vivo by: (1) developing and optimizing the anti-meso scFv vector. The avidity and the cytosolic signaling modules will be optimized to obtain highly efficient lentiviral vectors that retarget T cells to specifically kill tumor cells that express mesothelin at low effector to target ratios in vitro; (2) carrying out in vitro experiments to optimize the effector functions of anti-mesothelin scFv CD28-41BB-[unreadable]; T bodies. Experiments will determine optimal conditions for redirected T cell serial killing, cytokine production and proliferation, and compare this to natural MHC restricted CTLs; and (3) performing in vivo experiments in immunodeficient NOD/SCID/(32nu" mice xenografted with human tumors that express mesothelin. Here we will test the hypothesis that vectors with high affinity scFv receptors and 4-1BB and CD28 signaling modules will have the most potent anti-tumor effects. Finally, the engraftment, persistence and antitumor effects of chimeric T cells given by intravenous and intraperitoneal routes will be compared using bioluminescence imaging. In summary, we have assembled an outstanding team of basic and translational scientists to develop and test a universal T cell receptor to target some of the most common and drug resistant tumors. Lay Description. A common reason for failure of immunotherapy of epithelial tumors is that the immune system does not generate sufficient numbers of T cells to eradicate the tumor cells. It is now possible to use lentiviral vector technology to engineer T cells with potent and specific antitumor effects. In this project we will test engineered T cells that target mesothelin that is overexpressed on both uncommon tumors such as mesothelioma and a variety of commonly lethal tumors including pancreatic, ovarian and non-small cell lung carcinoma.